Pharmaceutical compositions for inhibiting angiogenesis comprising plant-derived natural compound

ABSTRACT

The present invention provides a pharmaceutical composition for inhibiting angiogenesis containing a plant-derived natural compound which can be effective for preventing or treating disorders or diseases associated with angiogenesis. The compound used as an active ingredient in the pharmaceutical composition of the present invention suppresses VEGF-induced angiogenic responses without cytotoxicity at a low concentration by inhibiting the expression of an anti-angiogenic factor (for example, VEGF), and thus remarkably improves the safety of a drug.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition forinhibiting angiogenesis comprising, as an active ingredient, aplant-derived natural compound having anti-angiogenic activity.

DESCRIPTION OF THE RELATED ART

Angiogenesis, the formation of new blood vessels from existingmicrovessels, is important in embryogenesis, wound healing, and tissueor organ regeneration [1,2]. However, pathological angiogenesis can leadto solid tumor growth and metastasis, diabetic retinopathy, and otherdiseases [3,4]. Accordingly, the inhibition of angiogenesis isconsidered a promising strategy for the treatment of cancer and otherhuman diseases linked with angiogenesis [2,5].

Natural compounds have played a positive role in the advancement of newbioactive small molecules as leads for drug development [6]. Somenatural compounds act as anti-viral, anti-bacterial, and anti-canceragents. For instance, etoposide, a topoisomerase inhibitor derived frompodophyllotoxin, a toxin found in the Podophyllum peltatum, prevents there-ligation of DNA strands. Accordingly, it is used as achemotherapeutic agent for the treatment of cancers such as Ewing'ssarcoma, lung cancer, testicular cancer, lymphoma, non-lymphocyticleukemia, and glioblastoma multiforme [7].

As part of our continuous efforts to discover new anti-angiogenic agentsfrom the natural plants, using cell-based screening, we screened 300crude extracts of natural plants for their effects on HUVECproliferation. We discovered that voacangine, a new natural smallmolecule, possesses anti-angiogenic properties. Voacangine(12-methoxyibogamine-18-carboxylic acid methyl ester), an indolealkaloid, was isolated from root bark of the Voacanga africana andTabernaemontana catharinensis trees (FIG. 1 a). A crude extract ofTabernaemontana catharinensis, which contained voacangine, was reportedto be a potent anti-cancer agent [8]. Voacangine has also been shown toinhibit capsaicin contraction in a dose-dependent manner [9]. However,there have been no reports demonstrating the anti-angiogenic activity ofthe compound. Here, we report for the first time that voacangine is anew natural small molecule that inhibits angiogenesis in vitro and invivo at a nontoxic dose.

Nicotine as one of main ingredients of tobacco has been reported as afactor for promoting angiogenesis, cancer cell proliferation andprogression of arteriosclerosis [16]. As described above, angiogenesiscauses growth and metastasis of solid tumors, diabetic retinopathy andsome diseases.

Based on properties of voacangine which has been described as activeingredients in anti-angiogenic pharmaceutical compositions comprisingplant-derived natural compounds filed in Korean Pat. Appln. No.10-2011-0124887, the present inventors have analyzed and verifiedregulation of nicotine-induced angiogenesis as well as potentials asinhibitors to nicotine-induced angiogenesis to suppress nicotine-inducedcancer cell proliferation and disease progression due to nicotinepoisoning.

Throughout this application, various patents and publications arereferenced and citations are provided in parentheses. The disclosure ofthese patents and publications in their entities are hereby incorporatedby references into this application in order to more fully describe thisinvention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

The present inventors have endeavored to develop an anti-angiogenicagent for effectively inhibiting angiogenesis without cytotoxicity. As aresult, the present inventors have discovered voacangine havinganti-angiogenic activity from Voacanga africana, and have found thatvoacangine may inhibit tumor cell-induced angiogenesis as well as VEGF-or hypoxia-induced angiogenesis, thereby being capable of being appliedto prevention or treatment of various angiogenesis-related diseases,disorders, or conditions.

Therefore, the present invention has been made in view of theabove-mentioned problems, and an aspect of the present invention is toprovide a pharmaceutical composition for inhibiting angiogenesiscontaining a plant-derived natural compound.

Other purposes and advantages of the present disclosure will becomeclarified by the following detailed description of invention, claims,and drawings.

Technical Solution

In accordance with an aspect of the present invention, there is provideda pharmaceutical composition for inhibiting angiogenesis, thecomposition comprising, as an active ingredient, a compound representedby Chemical Formula 1 below:

wherein, R₁, R₂, and R₃ each are independently hydrogen, C₁₋₁₂ alkyl,C₁₋₁₂ alkoxy, hydroxy, CN, CONH₂, halo, oxazolyl, C₁₋₁₂ alkylthio, ortrifluoro(C₁₋₂)alkyl.

The present inventors have endeavored to develop an anti-angiogenicagent for effectively inhibiting angiogenesis without cytotoxicity. As aresult, the present inventors have discovered voacangine havinganti-angiogenic activity from Voacanga africana, and have found thatvoacangine can inhibit tumor cell-induced angiogenesis as well as VEGF-or hypoxia-induced angiogenesis, thereby being capable of being appliedto prevention or treatment of various angiogenesis-related diseases,disorders, or conditions.

The composition of the present invention is expressed as “apharmaceutical composition for inhibiting angiogenesis”, which may beinterchangeably expressed as “a pharmaceutical composition forprevention or treatment of angiogenesis-related diseases” or “apharmaceutical composition for prevention or treatment of uncontrolledangiogenesis-related diseases”.

The compound used as an active ingredient in the pharmaceuticalcomposition of the present invention is represented by ChemicalFormula 1. In Chemical Formula 1 which defines the compound of thepresent invention, the term “C₁₋₁₂ alkyl” refers to straight chain orbranched chain saturated hydrocarbon group having 1-12 carbon atoms, andpreferably C₁- C₄ straight chain or branched chain alkyl, which is alower alkyl and includes methyl, ethyl, n-propyl, isopropyl, isobutyl,n-butyl, and t-butyl. The term “alkoxy” refers to —O— alkyl group. Whenthe compound is substituted with a substituted C₁-C₄ alkyl group, it issubstituted with halo-, preferably chloro-, and more preferablyfluoro-substituted alkyl group. The term “halo” refers to a halogengroup, which includes, for example, fluoro, chloro, bromo, and iodo, andis preferably fluoro, chloro, or bromo atom.

According to a preferable embodiment of the present invention, each R₁,R₂, and R₃ in Chemical Formula 1 is independently hydrogen, halo, orC₁₋₁₂ alkyl.

According to a preferable embodiment of the present invention, thecompound of the present invention is represented by Chemical Formula 2below:

According to a preferable embodiment of the present invention, thecompound of the present invention has activity of inhibiting theproliferation of human umbilical vascular endothelial cells (HUVECs).More preferably, the proliferation of the HUVECs is inhibited throughthe inhibition of expression of hypoxia-inducible factor-1α (HIF-1α) andits target gene, an anti-angiogenic factor (e.g., vascular endothelialgrowth factor (VEGF)).

According to a preferable embodiment of the present invention, thecompound of the present invention has activity of inhibiting tumorcell-induced angiogenesis, that is, hypoxia- or VEGF-inducedangiogenesis.

According to a preferable embodiment of the present invention, thecompound of the present invention has activity of inhibitingnicotine-induced angiogenesis.

Diseases, disorders or conditions that can be prevented or treated bythe pharmaceutical composition of the present invention include variousdiseases related to angiogenesis. Preferably, the pharmaceuticalcomposition of the present invention is utilized for prevention ortreatment of uncontrolled angiogenesis-related diseases or disordersincluding cancers, diabetic retinopathy, retinopathy of prematurity,corneal graft rejection, neovascular glaucoma, erythema, proliferativeretinopathy, psoriasis, hemophiliac joints, capillary proliferationwithin atherosclerotic plaques, keloids, wound granulation, vascularadhesions, rheumatoid arthritis, osteoarthritis, autoimmune diseases,Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, catscratch disease, ulcers, liver cirrhosis, glomerulonephritis, diabeticnephropathy, malignant nephrosclerosis, thrombotic microangiopathy,organ transplant rejection, glomerulopathy, diabetes, inflammation,neurodegenerative diseases, and nicotine addiction-related vesseldiseases.

According to a preferable embodiment of the present invention, thecancers capable of being prevented or treated by the composition of theinvention include, but are not limited to, brain cancer, neuroendocrinecancer, stomach cancer, lung cancer, breast cancer, ovarian cancer,liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer,pancreatic cancer, bladder cancer, adrenal cancer, colorectal cancer,colon cancer, cervical cancer, prostate cancer, bone cancer, skincancer, thyroid cancer, parathyroid cancer, and ureter cancer.

According to a preferable embodiment of the present invention, theautoimmune diseases capable of being prevented or treated by thecomposition of the present invention include, but are not limited to,alopecia greata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune adrenal disease, autoimmunehemolytic anemia, autoimmune hepatitis, autoimmune ovaritis andtestitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome, chronic inflammatory demyelinatingpolyradiculoneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid,CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Grave's disease, Guillain-Barre syndrome,Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathicthrombocytopenic purpuras, IgA nephropathy, juvenile arthritis, lichenplanus, lupus erythematosus, Meniere's disease, mixed connective tissuedisease, multiple sclerosis, type I or immune-mediated diabetes,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychondritis, autoimmune polyglandular syndrome, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoidarthritis, Sarcoidosis, scleroderma, stiff-person syndrome, systemiclupus erythematosus, lupus erythematosus, Takayasu's arteritis, temporalarteritis, giant cell arteritis, ulcerative colitis, uveitis, vitiligoand Wegener's granulomatosis.

According to a preferable embodiment of the present invention, theinflammatory diseases capable of being prevented or treated in thepresent invention may include: acute or chronic inflammatory disorders,such as inflammatory skin diseases (e.g., asthma, eczema, psoriasis,allergies, rheumatoid arthritis, psoriatic arthritis, atopic dermatitis,psoriasis, acne, atopic rhinitis (hay fever), allergic dermatitis(eczema), chronic sinusitis, or seborrheic dermatitis), bone diseases,gastritis, gout, gouty arthritis, ulcers, chronic bronchitis, acute lunginjury, lung inflammation, airway hypersensitivity, inflammatory boweldisease (e.g., Crohn's disease, ulcerative colitis), ankylosingspondylitis, sepsis, septic shock, vasculitis, and bursitis; autoimmunediseases, such as lupus, polymyalgia rheumatic, scleroderma, Wegener'sgranulomatosis, temporal arteritis, cryoglobulinemia, and multiplesclerosis; transplant rejection; cancers including solid tumors (e.g.,lung, CNS, intestine, kidney, and pancreas); Alzheimer's disease;atherosclerosis; viral infections (e.g., HIV or influenza); chronicviral infections (e.g., Epstein-Barr virus, cytomegalovirus, herpesvirus; and ataxia telangiectasia.

More preferably, the disease capable of being prevented or treated bythe pharmaceutical composition of the present invention is cancers,diabetic retinopathy, or proliferative retinopathy.

According to a preferable embodiment of the present invention, thecomposition of the present invention includes (a) a pharmaceuticallyeffective amount of the above-described compound of the presentinvention; and (b) a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically effective amount” refers toan amount enough to attain efficacy or activity of the above-describedcompound or a composition including the compound.

The pharmaceutical composition of the present invention includes apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier included in the pharmaceutical composition of the presentinvention is conventionally used in formulations, and examples thereofmay include, but are not limited to, lactose, dextrose, sucrose,sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate,gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, andmineral oil. The pharmaceutical composition of the present invention mayfurther include, besides the above components, a lubricant, a wettingagent, a sweetening agent, a flavoring agent, an emulsifier, asuspending agent, a preservative, and the like. Suitablepharmaceutically acceptable carriers and agents are described in detailin Remington's Pharmaceutical Sciences (19th ed., 1995).

The compound used as an active ingredient in the composition of thepresent invention may include, besides the compound of Chemical Formula1 itself, its pharmaceutically acceptable salt, hydrate, or solvate. Theterm “pharmaceutically acceptable salt” refers to a salt of the compoundof Chemical Formula 1, which contains a desired pharmacological effect,that is, activity of inhibiting tumor cell-induced angiogenesis.Examples of this salt are formed by using inorganic acids, such ashydrochloride, hydrobromide, and hydroiodide, and organic acids, such asacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,p-toluenesulfonate, bisulfate, sulfamate, sulfate, naphthylate,butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethane sulfonate, fumarate,glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,2-hydroxyethane sulfate, lactate, maleate, methane sulfonate,2-naphthalene sulfonate, nicotinate, oxalate, tosylate, and undecanoate.

The term “pharmaceutically acceptable hydrate” refers to a hydrate ofthe compound of Chemical Formula 1, which has a desired pharmacologicaleffect. The term “pharmaceutically acceptable solvate” refers to asolvate of the compound of Chemical Formula 1, which has a desiredpharmacological effect. The hydrate and solvate may be also prepared byusing the acids.

The pharmaceutical composition of the present invention may beadministered orally or parenterally. Examples of parenteraladministration may include intravenous, subcutaneous, intramuscular,intraperitoneal, transdermal injections, mucosal administration,administration of eye drops, and the like.

A suitable dose of the pharmaceutical composition of the presentinvention may be varied depending on factors, such as formulatingmethod, manner of administration, patient's age, body weight, sex, andmorbidity, food, time of administration, route of administration,excretion rate, and response sensitivity. Preferably, the dose of thepharmaceutical composition of the present invention is 0.001-100 mg/kg(body weight) in adults.

The pharmaceutical composition of the present invention may beformulated into a unit dosage form or injected in a multidose containerby using a pharmaceutically acceptable carrier and/or excipient,according to the method easily conducted by a person having ordinaryskills in the art to which the present invention pertains. Here, thedosage form may be a solution in an oily or aqueous medium, asuspension, a syrup, or an emulsion, an extract, a powder, a granule, atablet, or a capsule, and may further include a dispersant or astabilizer.

In accordance with another aspect of the present invention, there isprovided a method for inhibiting angiogenesis, the method includingadministering to a subject the composition of the present invention.

In accordance with still another aspect of the present invention, thereis provided a method for preventing or treating cancers, diabeticretinopathy, retinopathy of prematurity, corneal graft rejection,neovascular glaucoma, erythema, proliferative retinopathy, psoriasis,hemophiliac joints, capillary proliferation within atheroscleroticplaques, keloids, wound granulation, vascular adhesions, rheumatoidarthritis, osteoarthritis, autoimmune diseases, Crohn's disease,restenosis, atherosclerosis, intestinal adhesions, cat scratch disease,ulcers, liver cirrhosis, glomerulonephritis, diabetic nephropathy,malignant nephrosclerosis, thrombotic microangiopathy, organ transplantrejection, glomerulopathy, diabetes, inflammation, neurodegenerativediseases, or nicotine addiction-related vessel diseases, the methodincluding administering a subject the composition of the presentinvention.

Since the pharmaceutical composition and the compound as an activeingredient of the pharmaceutical composition, which are used in thepresent invention, are previously described, descriptions thereof areomitted to avoid excessive complication of the specification due torepetitive descriptions thereof.

Advantageous Effects

Features and advantages of the present invention are summarized asfollows:

(a) The present invention provides a pharmaceutical composition forinhibiting angiogenesis, capable of being effectively used forprevention or treatment of angiogenesis-related diseases or disorders.

(b) The compound used as an active ingredient in the pharmaceuticalcomposition of the present invention inhibits VEGF-induced angiogenicresponses without toxicity in low concentrations by inhibitingexpressions of angiogenic factors (e.g., VEGF), and thus significantlyimproves drug safety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-1 d represent chemical structure and anti-proliferativeactivity of voacangine on HUVECs. FIG. 1 a shows the chemical structureof voacangine (C₂₂H₂₈N₂O₃, MW 368.4). FIG. 1 b shows the effect of crudeextract of Voacanga africana on the proliferation of HUVECs. FIG. 1 cshows the effect of voacangine on cell proliferation. HUVECs weretreated with voacangine (1-20 μM) for 3 days, and cell growth wasmeasured using the MTT colorimetric assay. FIG. 1 d shows the effect ofvoacangine on cell viability. Cell viability was examined using thetrypan blue assay.

FIGS. 2 a-2 b represent anti-angiogenic activity of voacangine in vitro.Serum-starved HUVECs were stimulated by VEGF (30 ng/mL) in the presenceor absence of voacangine. FIG. 2 a shows the effect of voacangine on thetube forming ability of HUVECs. Arrows indicates broken tubes formed byVEGF-stimulated HUVECs. FIG. 2 b shows inhibitory activity of voacangineon endothelial cell invasion. The basal level capillary tube formation(a) and invasiveness (b) of HUVECs that remained in serum-free mediawere normalized to 100%.

FIG. 3 represents anti-angiogenesis activity of voacangine in vivo. (a)EtOH control, (b) RA (1 μg/egg), (c) voacangine (2 μg/egg), and (d)voacangine (4 μg/egg) were applied to the CAM, and the membrane wasobserved. Arrows indicate inhibition of neovascularization of CAM byvoacangine. Calculations were based on the proportion of positive eggsrelative to the total number of eggs tested.

FIGS. 4 a-4 c represent the effect of voacangine on the expression ofangiogenic factors. FIG. 4 a shows the expression level of HIF-1α andcyclin D1 were detected by Western blot. The level of tubulin was usedas an internal control. FIG. 4 b shows the expression level of VEGFprotein in HepG2 cells was determined by a VEGF immunoassay. FIG. 4 cshows tumor cell-induced angiogenesis. HUVECs were seeded in the upperchamber, and HepG2 was added to the lower chamber without VEGF. Nor,Normoxia; Hyp, Hypoxia.

FIGS. 5 a-5 b represent the inhibitory effect of voacangine onnicotine-induced angiogenesis in vitro. Serum-starved HUVECs werestimulated by nicotine (10 nM) in the presence or absence of voacangine.The nicotine treatment was analyzed to increase angiogenesis and cellinvasion activities by 2.5-fold and 2.0-fold, respectively,demonstrating that nicotine induces angiogenesis. Voacangine wasrevealed to inhibit nicotine-induced angiogenesis and invasion of HUVECsin a dose-dependent manner (FIGS. 5 a and 5 b). FIG. 5 a shows theeffect of voacangine on the tube forming ability of HUVECs. Arrowsindicates broken tubes formed by nicotine-stimulated HUVECs. FIG. 5 bshows inhibitory activity of voacangine on endothelial cell invasion.The basal level capillary tube formation (a) and invasiveness (b) ofHUVECs that remained in serum-free media were normalized to 100%.

PARTICULAR EMBODIMENTS OF THE INVENTION

The present invention will now be described in further detail byexamples. It would be obvious to those skilled in the art that theseexamples are intended to be more concretely illustrative and the scopeof the present invention as set forth in the appended claims is notlimited to or by the examples.

EXAMPLES Materials and Methods

Materials

Voacangine (12-methoxyibogamine-18-carboxylic acid methyl ester) waspurchased from THC Pharm (Frankfurt, Germany). Endothelial growthmedium-2 (EGM-2) was purchased from Lonza (Walkersville, Md.). RPMI 1640and fetal bovine serum (FBS) were purchased from Invitrogen (GrandIsland, N.Y.). Vascular endothelial growth factor (VEGF), Matrigel andTranswell chamber systems were obtained from KOMA Biotech (Seoul,Korea), BD Bioscience (Bedford, Mass.) and Corning Costar (Corning,N.Y.), respectively. Anti-HIF-1α, anti-cyclin D1 and anti-tubulinantibody were purchased from BD Bioscience, Cell Signaling (Beverly,Mass.) and Millipore (Billerica, Mass.), respectively.

Cell Culture and Proliferation Assay

Human umbilical vascular endothelial cells (HUVECs) were grown for 7-11passages in EGM-2 medium supplemented with 100 FBS. HepG2 (human livercarcinoma) cells were grown in RPMI 1640 containing 10% FBS and 1%antibiotics. All cell lines were maintained at 37° C. in a humidified 5%CO₂ incubator. Cell proliferation was measured using a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)colorimetric assay, and cell viability was assessed using Trypan bluestaining [10].

Capillary Tube Formation Assay

Matrigel (10 mg/mL) was used to coat a 48-well plate and allowed topolymerize for 1 h at 37° C. HUVECs (6×10⁴ cells) were seeded on thesurface of the Matrigel, and then test compounds were added for 4-16 hat 37° C. in the presence or absence of VEGF (30 ng/mL). Morphologicalchanges in the cells and formation of tubular structures were observedunder a microscope (IX71, Olympus) and photographed at 100×magnification (DP70, Olympus) [11].

Chemoinvasion Assay

To examine the invasiveness of HUVECs in vitro, we used a Transwellchamber system with 8.0-μm pore polycarbonate filter inserts [12].Briefly, the lower side of the filter was coated with gelatin (10 μL, 1mg/mL) and the upper side was coated with Matrigel (10 μL, 3 mg/mL).Test compounds were added to the lower chamber in the presence of VEGF(30 ng/mL), and HUVECs (7×10⁵ cells) were placed in the upper chamber ofthe filter. The chamber was incubated at 37° C. for 18 h, and then thecells were fixed with 70% methanol and stained with hematoxylin andeosin. The invasiveness of cells was measured by counting the number ofwhole cells in the lower side of the filter using a microscope at 100×magnification, and cells were photographed at 100× magnification.

Chorioallantoic Membrane (CAM) Assay

The CAM assay was performed as described previously [13]. Fertilizedchicken eggs were kept in a humidified incubator at 37° C. for 3 days.Approximately 2-3 mL of egg albumin was removed with a hypodermicneedle, allowing the CAM and yolk sac to drop away from the shellmembrane. On day 5, a 2.5-cm diameter window was made with a razor andtweezers, and a compound-loaded Thermanox coverslip (NUNC, Rochester,N.Y.) was applied to the CAM surface. After further incubation for 2days, 2-3 mL of Intralipose (Greencross Co, Suwon, Korea) was injectedbeneath the CAM and the membrane was observed under a microscope.Retinoic acid (RA) was used as a positive control.

Western Blot Analysis and Hypoxic Conditions

The cell lysates were separated by 10% SDS-PAGE, followed by transfer toPVDF membranes (Millipore, Bedford, Mass.) using standardelectroblotting procedures. Blots were then blocked and immunolabeledovernight at 4° C. with primary antibodies, including anti-HIF-1α andanti-tubulin antibodies. Immunolabeling was detected by an enhancedchemiluminescence (ECL) kit (GE Healthcare, Buckinghamshire, UK)according to the manufacturer's instructions. For hypoxic conditions,cells were incubated at 5% CO₂ with 1% O₂ balanced with N₂ in ananaerobic chamber (Forma).

Measurement of VEGF by ELISA

The VEGF concentration in media from voacangine-treated cells wasdetermined using a VEGF Immunoassay kit (R&D Systems, Minneapolis,Minn.) according to the manufacturer's instructions. The results wereexpressed as concentration of VEGF relative to the total amount of VEGFfrom each well.

In Vitro Tumor Cell-Induced Angiogenesis Assay (Combinated-ChemoinvasionAssay)

To examine the invasive activity of HUVEC-induced tumor cells, aTranswell chamber system with 8.0-μm pore polycarbonate filter insertswas used. Briefly, the lower side of the filter was coated with gelatin(10 μL, 1 mg/mL) and the upper side was coated with Matrigel (10 μL, 3mg/mL). Next, tumor cell HepG2 was added to the lower chamber. Testcompounds were added to the lower chamber without VEGF and HUVECs (7×10⁵cells) were placed in the upper chamber of the filter. The chamber wasincubated at 37° C. for 18 h, and then the cells were fixed with 70%methanol and stained with hematoxylin and eosin. Invasiveness wasmeasured by counting the number of whole cells on the lower side of thefilter using a microscope at 100×magnification, and cells werephotographed at 100× magnification [14].

Statistical Analysis

Results are expressed as the mean ±standard error (SE). Student's t-testwas used to determine the statistical significance between control andtest groups. A p-value less than 0.05 was considered statisticallysignificant.

Results and Discussion Voacangine Potently Inhibits the Proliferation ofHUVECs

We found that the crude extract of Voacanga africana inhibits theproliferation of HUVECs in a dose-dependent manner (FIG. 1 b).Voacangine is a known principal component of this extract. Therefore, weinvestigated whether voacangine is responsible for the observedanti-proliferative activity of the extract. As shown in FIG. 1C,voacangine inhibited cell growth at 10 μM. Notably, it exerted a greatergrowth inhibition effect on HUVECs than on other normal and cancer celllines (Table 1).

TABLE 1 IC₅₀ values of voacangine on various cell lines Normal cellsCancer cells Cell lines HUVECs CHANG HeLa HT1080 HepG2 IC₅₀ (μM) 18 2623 33 42

To determine the optimum dose of voacangine without cytotoxic sideeffects, various concentrations of voacangine (1-20 μM) were applied toHUVECs, and cell viability was determined using the trypan blueexclusion method. Voacangine exhibited no cytotoxicity on HUVECs atdoses up to 20 μM for 3 days. Accordingly, the following studies wereperformed using a concentration range of 10-20 μM (FIG. 1 d).

Voacangine Showed Anti-Angiogenic Activity In Vitro and In Vivo

We next investigated the effect of voacangine on the angiogenicphenotypes of HUVECs in vitro, such as tube formation and chemoinvasion.Serum-starved HUVECs were stimulated by VEGF with or without voacangine.As shown FIG. 2 a, voacangine inhibited VEGF-induced tube formation in adose-dependent manner with no cytotoxic effects. The effect ofvoacangine on the invasive activity of HUVECs induced by VEGF was alsoinvestigated. Voacangine inhibited the VEGF-induced enhancedinvasiveness of HUVECs in a dose-dependent manner (FIG. 2 b). These dataindicate that voacangine effectively inhibits VEGF-induced angiogenesisin vitro.

The anti-angiogenic activity of voacangine was further validated in vivoby using a chick embryo chorioallantoic membrane (CAM) assay. Aftertreatment with voacangine for 2 days, the CAM was observed under amicroscope. Normally, developed CAMs exhibit an extensive capillarynetwork. However, voacangine dose-dependently inhibited capillaryformation during CAM development with no apparent signs of thrombosis orhemorrhage (FIG. 3). These results demonstrate that voacangine potentlyinhibits angiogenesis both in vitro and in vivo without cytotoxiceffects.

Voacangine Inhibits Tumor Cell-Induced Angiogenesis

Hypoxia-inducible factor-1α (HIF-1α) plays a key role in tumorangiogenesis by regulating the expression of angiogenic factors,including VEGF [15]. HIF-1α overexpression has been implicated in manyhuman cancers. Thus, we examined the effect of voacangine on HIF-1αexpression levels under hypoxic conditions. The expression level ofHIF-1α in human hepatocellular carcinoma (HepG2) cells during hypoxiawas dose-dependently reduced by voacangine without inhibiting thesynthesis of other proteins related to the cell cycle (cyclin D1) andcytoskeleton (tubulin) (FIG. 4 a).

As the result of reduced HIF-1α expression, voacangine treatmentinhibited the hypoxia-induced expression of VEGF, a HIF-1α target gene,in a dose-dependent manner (FIG. 4 b). Moreover, tumor cell-inducedinvasiveness of HUVECs by hypoxia was dose-dependently inhibited byvoacangine (FIG. 4 c). These results demonstrate that voacanginepotently inhibits tumor cell-induced angiogenesis through suppression ofthe angiogenic factor, HIF-1α, with no observed cytotoxic effects.

In this study, our results clearly demonstrate that voacangine, anactive principal component of Voacanga africana extract, exhibitsanti-angiogenic activity in vitro and in vivo. In HUVECs, the expressionlevels of HIF-1α and its target gene, VEGF, were dose-dependentlysuppressed by voacangine. In addition, voacangine inhibited tumorcell-induced invasiveness in a dose-dependent manner. Overall, theseresults suggest that this compound might provide the basis for thedevelopment of novel anti-angiogenic agents. It is noteworthy thatvoacangine effectively suppresses VEGF- and hypoxia-induced angiogenesisat lower doses than are necessary to inhibit HUVEC growth, suggestingthat the compound may specifically perturb angiogenic signalingpathways. Moreover, the unique chemical structure of voacangine (with aniboga alkaloid as a core moiety) may provide new insights into themechanisms underlying angiogenesis signaling pathways. Furtherinvestigation identifying and validating the targets of the small,naturally occurring molecule voacangine will help to decipher theinteresting anti-angiogenic mechanisms of the compound and open a newgate into angiogenesis biology.

Having described a preferred embodiment of the present invention, it isto be understood that variants and modifications thereof falling withinthe spirit of the invention may become apparent to those skilled in thisart, and the scope of this invention is to be determined by appendedclaims and their equivalents.

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1-13. (canceled)
 14. A method for inhibiting angiogenesis, the methodcomprising administering to a subject a composition comprising as anactive ingredient a compound represented by Chemical Formula 1 below:

wherein, R₁, R₂, and R₃ each are independently hydrogen, C₁₋₁₂ alkyl,C₁₋₁₂ alkoxy, hydroxy, CN, CONH₂, halo, oxazolyl, C₁₋₁₂ alkylthio, ortrifluoro(C₁₋₂)alkyl.
 15. The method of claim 14, wherein R₁, R₂, and R₃each are independently hydrogen, halo, or C₁₋₁₂ alkyl.
 16. The method ofclaim 14, wherein the compound inhibits the proliferation of humanumbilical vascular endothelial cells (HUVECs).
 17. The method of claim16, wherein the proliferation of HUVECs is inhibited through theinhibition of hypoxia-inducible factor-1α (HIF-1α) expression.
 18. Themethod of claim 14, wherein the compound inhibits tumor cell-inducedangiogenesis.
 19. The method of claim 14, wherein the compound inhibitshypoxia- or vascular endothelial growth factor (VEGF)-inducedangiogenesis.
 20. The method of claim 14, wherein the compound inhibitsnicotine-induced angiogenesis.
 21. The method of claim 14, wherein thecomposition inhibits the expression of angiogenic factors.
 22. Themethod of claim 21, wherein angiogenic factors include VEGF.
 23. Themethod of claim 14, wherein the composition is used for prevention ortreatment of cancers, diabetic retinopathy, retinopathy of prematurity,corneal graft rejection, neovascular glaucoma, erythema, proliferativeretinopathy, psoriasis, hemophiliac joints, capillary proliferationwithin atherosclerotic plaques, keloids, wound granulation, vascularadhesions, rheumatoid arthritis, osteoarthritis, autoimmune diseases,Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, catscratch disease, ulcers, liver cirrhosis, glomerulonephritis, diabeticnephropathy, malignant nephrosclerosis, thrombotic microangiopathy,organ transplant rejection, glomerulopathy, diabetes, inflammation,neurodegenerative diseases, or nicotine addiction-related vesseldiseases.
 24. The method of claim 14, wherein the compound isrepresented by Chemical Formula 2 below:


25. A method for preventing or treating cancers, diabetic retinopathy,retinopathy of prematurity, corneal graft rejection, neovascularglaucoma, erythema, proliferative retinopathy, psoriasis, hemophiliacjoints, capillary proliferation within atherosclerotic plaques, keloids,wound granulation, vascular adhesions, rheumatoid arthritis,osteoarthritis, autoimmune diseases, Crohn's disease, restenosis,atherosclerosis, intestinal adhesions, cat scratch disease, ulcers,liver cirrhosis, glomerulonephritis, diabetic nephropathy, malignantnephrosclerosis, thrombotic microangiopathy, organ transplant rejection,glomerulopathy, diabetes, inflammation, neurodegenerative diseases, ornicotine addiction-related vessel diseases, the method comprisingadministering a subject a composition comprising as an active ingredienta compound represented by Chemical Formula 1 below:

wherein, R₁, R₂, and R₃ each are independently hydrogen, C₁₋₁₂ alkyl,C₁₋₁₂ alkoxy, hydroxy, CN, CONH₂, halo, oxazolyl, C₁₋₁₂ alkylthio, ortrifluoro(C₁₋₂)alkyl.
 26. The method of claim 25, wherein R₁, R₂, and R₃each are independently hydrogen, halo, or C₁₋₁₂ alkyl.
 27. The method ofclaim 25, wherein the compound is represented by Chemical Formula 2below: